Severe Acute Respiratory Syndrome Coronavirus Replication Inhibitor That Interferes with the Nucleic Acid Unwinding of the Viral Helicase

Abstract

Severe acute respiratory syndrome (SARS) is a highly contagious disease, caused by SARS coronavirus (SARS-CoV), for which there are no approved treatments. We report the discovery of a potent inhibitor of SARS-CoV that blocks replication by inhibiting the unwinding activity of the SARS-CoV helicase (nsp13). We used a Förster resonance energy transfer (FRET)-based helicase assay to screen the Maybridge Hitfinder chemical library. We identified and validated a compound (SSYA10-001) that specifically blocks the double-stranded RNA (dsRNA) and dsDNA unwinding activities of nsp13, with 50% inhibitory concentrations (IC 50 s) of 5.70 and 5.30 M, respectively. This compound also has inhibitory activity (50% effective concentration [EC 50 ] ‫؍‬ 8.95 M) in a SARS-CoV replicon assay, with low cytotoxicity (50% cytotoxic concentration [CC 50 ] ‫؍‬ >250 M), suggesting that the helicase plays a still unidentified critical role in the SARS-CoV life cycle. Enzyme kinetic studies on the mechanism of nsp13 inhibition revealed that SSYA10-001 acts as a noncompetitive inhibitor of nsp13 with respect to nucleic acid and ATP substrates. Moreover, SSYA10-001 does not affect ATP hydrolysis or nsp13 binding to the nucleic acid substrate. SSYA10-001 did not inhibit hepatitis C virus (HCV) helicase, other bacterial and viral RNA-dependent RNA polymerases, or reverse transcriptase. These results suggest that SSYA10-001 specifically blocks nsp13 through a novel mechanism and is less likely to interfere with the functions of cellular enzymes that process nucleic acids or ATP. Hence, it is possible that SSYA10-001 inhibits unwinding by nsp13 by affecting conformational changes during the course of the reaction or translocation on the nucleic acid. SSYA10-001 will be a valuable tool for studying the specific role of nsp13 in the SARS-CoV life cycle, which could be a model for other nidoviruses and also a candidate for further development as a SARS antiviral target. Severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for the life-threatening viral respiratory illness known as SARS, which emerged from Southern China in November 2002 and spread to other parts of the world, including North America, South America, and Europe (50, 64). There is currently no approved therapeutic agent for the treatment of SARS-CoV infections. Although SARS currently does not pose a public health threat, the likelihood of future occurrences of both SARS-CoV and related viruses necessitates continuous research for identification of antiviral therapies.SARS-CoV contains a single-stranded, 5=-capped, polyadenylated positive-strand RNA genome that is ϳ29.7 kb long (40,45). The first open reading frame (ORF1a/b) encompasses about twothirds of the genome and codes for the replicase proteins (41). Following a Ϫ1 frameshift signal, translation continues in ORF1b after initiation at ORF1a. The virally encoded chymotrypsin-like protease 3CLpro (also called M pro or main protease) and the papain-like protease (PLP) cleave (by autoproteolysis) the newly form...

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